Cultivated rice, which primarily includes Oryza sativa (Asian cultivated rice) and Oryza glaberrima (African cultivated rice), contains 22 wild species that are not cultivated (Solis et al., 2020). O. sativa is cultivated worldwide, whereas O. glaberrima, is predominantly grown in Africa. Wild rice ancestors have adapted to various geographically distinct habitats (Atwell et al., 2014). The 22 wild ancestors constituted the largest gene pool. Oryza species are highly variable and comprise 11 distinct genomes, including six diploids (AA, BB, CC, EE, FF, and GG) and five allotetraploids (BBCC, CCDD, KKLL, HHJJ, and HHKK) (Stein et al., 2018). They differ in morphological characteristics such as growth habit, plant height, flowering, leaf size, panicle size, and branching, awning, and seed size.

Wild relatives of rice are grouped into three gene pools (primary, secondary, and tertiary) based on their ease of hybridization with cultivated rice and phylogenetic relationships (Harlan and de Wet, 1971). The primary gene pool comprises the O. sativa complex (AA genome) with close relatives of cross-compatible rice and a secondary gene pool consisting of O. officinalis complex (BB to FF genomes) with less closely related species and a tertiary gene pool constituted with O. meyeriana complex, O. ridleyi complex, and O. schlechteria complex (GG to KKLL genomes) with more distant relatives of rice, necessitating embryo rescue, chromosome doubling, or bridging species to facilitate gene transfer (Solis et al., 2020) ( Figure 1 ).

Studies have revealed that indigenous species of rice could potentially be used as genetic resources for abiotic stresses such as submergence (Luo et al., 2020; Sasayama et al., 2022) drought (Hamzelou et al., 2020; Anilkumar et al., 2023), heat (Prasanth et al., 2012; Khan et al., 2019; Jagadish et al., 2021), etc. However, few limited published studies have reported the significance of wild rice species in elevating salinity tolerance in cultivars (Solis et al., 2020).

Indeed, very few review articles have focused on the genetic enhancement of salinity tolerance in wild O. sativa species. Hence, in this manuscript, we have reviewed the impact of salinity effects in rice, the importance of wild rice species in breeding for salinity tolerance, characterization and mechanisms of tolerance in wild rice, deployment of wild rice genes in rice breeding, and challenges and opportunities for the incorporation of wild gene loci to develop salt-resilient rice cultivars.

Wild progenitors adapted to various changing climatic conditions display tremendous genetic diversity and carry genetic loci associated with unique agronomic and adaptive traits (Hellwig et al., 2021). The basic requirement of a successful crop breeding program is the availability and accessibility of genetic variability in target traits (Perrino and Perrino, 2020). When there is exhaustion in genetic variation among domesticated genotypes, breeders are left with access the diversity available in land races and wild ancestors as alternate sources of variability.

Asian cultivated rice has evolved from its wild ancestor Oryza rufipogon through human efforts (Wang et al., 1992). Similarly, the African rice O. glaberrima was domesticated from the African wild progenitor Oryza barthii. Erect growth, non-shattering, and increased grain number and weight were targeted during selective breeding. The intense selection and breeding that occurred during domestication could have probably resulted in the loss of genetic variability and the loci associated with stress tolerance in cultivars. O. sativa represents <20% of the total variation compared to that found in various wild species (Stein et al., 2018), and significant natural variability for salt tolerance still exists in wild species (Munns et al., 2016), which can be exploited to improve cultivated rice. There are certain wild species such as Oryza coarctata, a distantly related wild rice that thrives under extreme salinity (450 mM NaCl). However, the crossability barrier limits their utility.

Owing to tall plant stature, photosensitivity, poor grain type, low seed set, high grain shattering, and low yield potential, natural wild accessions are regarded as poor agronomic performers (Sanchez et al., 2013). Despite this, the recovery of widely adaptable cultivars to diverse challenging environments would be higher when wild relatives of rice are used in the crossing program (Jin et al., 2021). Insights into morphological and physiological responses to salt stress are crucial for exploiting stress tolerance through distant crosses. The extent to which wild rice progenitors demonstrate tolerance and the probable genetic defense strategies behind tolerance remains partially understood. Crossing barriers, linkage drag, and epistatic effects from unadapted wild genes when introgressed into elite cultivars may complicate mainstream breeding. However, advances in genomic tools and techniques, particularly embryo rescue techniques and deploying molecular marker-based advanced backcrosses, identification, selection, and incorporation of target quantitative trait loci (QTLs) into elite varieties, could successfully generate improved versions of cultivars (Dai et al., 2022).

The genetic wealth of rice diversity was systematically preserved in gene banks. As of December 2023 (URL-https://www.irri.org/international-rice-genebank), the International Rice Gene Bank Collection Information System (IRGCIS) of the International Rice Research Institute (IRRI) documented 1,32,000 accessions of both cultivated and wild rice. Nevertheless, only a few wild accessions have been evaluated using various salt-screening methods (Kumar et al., 2015). The primary gene pool consisting of cultivated rice species (O. sativa and O. glaberrima) and wild rice species (Oryza nivara, O. rufipogon, O. barthii, Oryza longistaminata, Oryza meridionalis, and Oryza glumaepatula) are analyzed for detecting tolerant wild accessions in limited research investigations. A large untapped genetic diversity is available in the secondary and tertiary gene pools, which are additional sources of new salinity tolerance genes. These gene pools can be used to introduce beneficial genes into superior rice varieties (Prusty et al., 2018; Solis et al., 2020).

Over the past three decades, there has been substantial advancement in the genetic diversity analysis of wild rice resources and the selection of suitable donor parents. Following the standard Yoshida solution culture method (Gregorio et al., 1997) and the modified Yoshida solution culture method (Singh et al., 2010), several germplasm accessions were screened at the seedling stage to identify the donors. A few accessions of O. rufipogon and O. nivara that survived salinity levels as high as 12 dSm⁻¹ (Habiba et al., 2015) were identified. O. rufipogon is considered the best donor among the closest wild relatives belonging to the AA genome, followed by the distant wild relatives O. coarctata, O. latifolia, and Oryza alta (Solis et al., 2020).

At the ICAR-National Institute of Plant Biotechnology, India, 800 accessions of O. nivara and O. rufipogon collected across ecologies were evaluated (Singh et al., 2018). The accession NKSWR 173 recorded high seedling stage salt tolerance (150 mM NaCl) (Mishra et al., 2016). A survey of 22 accessions of wild species screened under high salinity (240 mM NaCl) in a hydroponic system revealed seven accessions, Oryza minuta, Oryza grandiglumis, Oryza latifolia, O. alta, Oryza rhizomatis, O. coarctata, and Oryza eichingeri, with higher levels of tissue tolerance and chlorophyll preservation in leaves as compared to the donor parents for salinity tolerance(Pokkali, Nona Bokra, and FL478) and salt-sensitive checks (IR29 and IR75862-206-2-8-3) (Prusty et al., 2018). Mondal et al. (2018) and Mishra et al. (2022) studied the halophytic wild species O. coarctata abundant in Indian coastal regions as a source of salinity tolerance genes.

In the past two decades, genome sequencing technology has improved the utilization of genetic variation in wild Oryza species for crop improvement. Efforts to sequence wild rice genomes began in 2003 with the establishment of the International Oryza Map Alignment Project (IOMAP). It has provided an in-depth characterization of wild rice genomes to discover and exploit genes/genomic regions governing diverse traits for their transfer into cultivated rice (Wing et al., 2005). Stein et al. (2018) sequenced genomes of seven wild species (O. rufipogon, O. nivara, O. barthii, O. glumaepatula, O. meridionalis, O. punctata, and L. perrieri) and deposited at National Center for Biotechnology Information (NCBI), Bethesda, Maryland and provided an array of diversity panels. The genome of O. coarctata has been sequenced at ICAR-NIPB, India, which would be a valuable addition to the I-OMAP project and would supplement the current genomic resources of wild and cultivated species (Mondal et al., 2018). This provides insight into the genetic makeup of O. coarctata and broadens the gene pool for the enhancement of cultivated rice. Multiple studies have employed whole genome sequencing and resequencing information from cultivated and wild rice population species to identify genomic regions harboring improved agronomic traits as well as adaptive traits, such as resistance to biotic stresses and tolerance to abiotic stresses, including salinity tolerance (Huang et al., 2012; Xu et al., 2012).

Rice is the main nutritional source for more than half of the world’s population (Zhou et al., 2020). Globally, rice is the third-largest cereal grown in 162 million hectares, with an annual production of 755 million tons (FAO, 2022). Soil salinity and alkalinity are the leading abiotic stressors in coastal and inland areas, followed by drought (Islam et al., 2021). Even moderate salt stress has been reported to reduce the rice yield by 68% (Naheed et al., 2007). The changing global climate is expected to cause a significant increase in soil salinization owing to inadequate rainfall, high evaporation rates, and seawater intrusion (Cheng et al., 2020).

Salt-affected soils are broadly classified as sodic–alkaline, saline, or saline–sodic soils (Eynard et al., 2005). In alkaline soils, carbonates and bicarbonates of sodium and magnesium are the most prevalent anions, whereas chlorides and sulfates of sodium and magnesium are frequently found in saline soils. Sodic soils have pH >8.5, the electrical conductivity of saturation extract (ECe) <4 dS m⁻¹ and exchangeable sodium percentage (ESP) >15. Saline soils near the coastal regions, ECe >4 dS m⁻¹, pH < 8.5, and ESP <15. Saline sodic soils exhibit saline and sodic characteristics, such as variable pH, ECe ≥4 dS m⁻¹, and ESP ≥15%.

Plants are categorized into halophytes and glycophytes based on their responses to salt stress, (Flowers and Colmer, 2008; Mishra and Tanna, 2017). Halophytes thrive in highly saline soils. Most of the crops are salt-sensitive glycophytes. Rice has a threshold salt concentration of >30 mM NaCl (ECe = 3 dS m⁻¹) (USDA et al., 2008). A decrease in yield of 12% was reported for each unit, exceeding the specified threshold (Reddy et al., 2014). Excessive salinity causes osmotic stress and ion toxicity in crop plants. Osmotic pressure reduces soil osmotic potential and causes decreased water uptake, further inhibiting stomatal opening, photosynthesis, elongation, and cell proliferation. This results in a slower growth. Ionic stress causes rapid accumulation of toxic Na⁺ and Cl⁻, disrupting metabolic processes, resulting in early senescence and reduced stomatal conductance, leading to decreased photosynthesis, biomass, and poor yield (Yu et al., 2017). Salinity stress, apart from reducing germination, causes whitening of affected leaf tips, leaf rolling, stunted plant growth, patchy appearance in the field, reduced tillering, delayed panicle emergence, length and the number of panicles, reduces pollen viability, spikelet fertility, spikelet number/panicle, and ultimately grain yield, and in cases of increased severity, results in the death of the rice plant ( Figure 2 ).

Plants adapt to high salt through various physiological and biochemical defense systems namely high initial seedling vigor, early maturity, sodium exclusion, sodium compartmentalization in vacuoles of roots and older leaves, osmotic adjustment, control of Reactive Oxygen Species (ROS) through production of antioxidants, and programmed cell death.

During the domestication of cultivated rice from local landraces and traditional varieties, selection pressure for productivity traits favored a few genes at the expense of many others. Hence, domesticated varieties have less genetic diversity for other non-selected traits, such as biotic and abiotic stresses, including salinity tolerance, compared to wild species and landraces. This implies that the cultivated gene pool has a narrow genetic base; hence, further gain in salinity tolerance is difficult to achieve. Consequently, the developed cultivars were found to be salt sensitive or moderately tolerant to salinity. Interestingly, wild rice gene pools possess extensive genetic diversity as they grow in undisturbed natural habitats. This diversity can enrich the cultivated gene pool with higher salt tolerance through introgression of wild genes. Salt stress profoundly affects various morphological, physiological, and biochemical processes in rice plants ( Table 1 ).

Plants have evolved protective mechanisms at the cellular, organellar, and whole plant levels to recover from salinity stress. Rice cultivars exhibit genetic variation in their adaptive strategies against salinity (Khan et al., 2020). The crop has developed a series of adaptive mechanisms, including (i) osmotic adjustment, (ii) compartmentation and ion homeostasis, (iii) antioxidant defense, and (iv) programmed cell death ( Figure 3 ).

Wild species have evolved stronger morphological, physiological, genetic, and molecular adaptive mechanisms than cultivars have. They exhibit sodium secretion through special structures such as salt glands, improved ion homeostasis in roots and shoots, increased osmolyte biosynthesis, strong tissue tolerance, greater detoxification of ROS, and enhanced osmotic tolerance compared to cultivars. Several wild rice donors with increased tolerance to salinity have been, i.e., O. coarctata (Majee et al., 2004; Sengupta and Majumder, 2010; Garg et al., 2014), O. punctata, O. officinalis (Prusty et al., 2018), and O. rufipogon (Tian et al., 2011; Zhou et al., 2016).

Nakamura et al. (2002) reported higher seedling survival and high photosynthetic rates in O. latifolia and O. rufipogon up to 300 mM NaCl concentration than cultivated salt tolerant SR26-B and salt sensitive IR28. O. coarctata, earlier known as Porteresia coarctata, an allotetraploid wild rice halophyte with KKLL genome (2n = 4x = 48), is the only distant rice which could withstand extreme salinity (500 mM to 650 mM NaCl) as reported by Sengupta et al. (2008). It is a unique wild rice that grows in mangroves along coastal belts. It exhibits multiple defense mechanisms of tolerance and mainly adopts a salt excretion strategy to reduce the high salt load in photosynthetic tissues (Sengupta and Majumder, 2010), thereby retaining a high photosynthetic rate. It contains characteristic hairs known as trichomes on the upper surface of the leaves, through which sodium and chloride salts are excreted (Prusty et al., 2018). It deploys vacuolar Na⁺ compartmentation mediated by Na⁺ transporters (OcNHX1, OcSOS1, OcHKT1;4, and OcHKT1;5) to maintain a low cytosolic Na⁺/K⁺ ratio in the leaf mesophyll, even though it continues to grow in saline water. Despite high salinity, it sustains a low water content (Senthilkumar et al., 2005). Additionally, it possesses a greater Na⁺ and K⁺ retention capacity in shoots than in roots and eliminates ROS through enzymatic processes (Sengupta and Majumder, 2009).

A study conducted by Platten et al. (2013) using 103 O. sativa and 12 O. glaberrima accessions indicated the operation of ion homeostasis mechanisms in O. glaberrima through salt accumulation in leaves, which was independent of OsHKT1;5, as observed in O. sativa. Pushpalatha et al. (2013) evaluated 15 O. nivara and O. rufipogon introgression lines in the background of KMR3 and Swarna cultivars at various salinity levels (0 mM to 200 mM NaCl) during germination, vegetative, and reproductive growth stages and study revealed a Na⁺ exclusion mechanism and osmoprotection by proline synthesis in KMR3 ILs (K463 and K478), vacuolar sodium sequestration, and consistent chlorophyll content in Swarna ILs (S166, S3-1, S14, and S75) in response to salt stress.

Nishizawa et al. (2015), (Nishizawa et al., 2016) reported similar constant photosynthetic rates and sodium accumulation in O. officinalis and O. latifolia accessions. However, a relationship between stomatal conductance and net photosynthetic rate could not be demonstrated in O. latifolia compared to O. officinalis, indicating the possibility of the presence of a new set of salinity tolerance loci. O. rufipogon and O. nivara have been reported to possess a pool of genes that maintain K⁺ homeostasis, Na⁺ exclusion, and sodium compartmentalization (Ganeshan et al., 2016; Tian et al., 2017). Another wild relative, O. australiensis recorded proline accumulation, low Na⁺ content, and a low Na⁺/K⁺ ratio in the shoots and roots (Yichie et al., 2018).

Prusty et al. (2018) investigated 22 wild species in a hydroponic experiment along with four cultivated tolerant checks (Nona Bokra, Pokkali, and FL478) and two sensitive checks (IR 29 and IR 75862-206-2-8-3) under high saline stress (240 mM NaCl). Two wild species, O. latifolia and O. alta, survived up to 26 days to 33 days, and O. coarctata grew without deleterious effects. Interestingly, O. eichingeri, O. minuta, and O. coarctata accumulated high Na⁺ content in their roots and had low oxidative damage. Gene expression studies suggested the involvement of the OsHKT1;4 transporter gene for mediating Na+ exclusion in leaves, but Na compartmentation occurred independently of the tonoplast-localized OsNHX1 transporter gene. Shahzad et al. (2022) attributed the tolerance in wild rice genotypes of O. alta and O. barthii, to a greater tissue tolerance mechanism. Orthologous alleles of the stress-responsive VOZ gene from wild species, viz., O. brachyantha, O. longistaminata, and O. nivara, could act as potential donors for salinity stress improvement (Ganie et al., 2020). Nan et al. (2020) identified activation of OrWRKY genes in O. rufipogon under salt stress.

Nguyen (2022) investigated physiological traits governing salt tolerance in 18 Australian wild rice accessions of O. australiensis, O. rufipogon, and O. meridionalis under high salinity (200 mM NaCl) together with three cultivars, namely IR 29, salt sensitive and Pokkali, salt tolerant. O. australiensis accessions displayed high net photosynthesis, high relative water content, and low Na⁺ and Na⁺/K⁺ in the shoots and roots. Gene expression analysis revealed upregulation of proline synthesis genes OsP5CS1 and OsP5C2, and downregulation of the proline degradation gene OsProDH. Thus, osmoregulation and ion homeostasis are the key tolerance mechanisms in O. australiensis accessions. Solis et al. (2022) demonstrated higher Na⁺ uptake and reduced Na⁺ effluxes in O. alta, O. latifolia, and O. coarctata. The expression of NHX1 and SOS1/NHX7 genes that govern tissue tolerance triggered by salt stress. Detailed information on the candidate genes and their mechanisms are listed in Table 2 .

Adaptive mechanisms may occur singly or in combination, depending on the species and growth stage of the crop. Different accessions of the same wild Oryza species could have a distinct genetic basis for phenotypic expression. With better knowledge of the mechanisms in wild species, appropriate breeding methods could be formulated to enhance yield under salt stress than what is currently obtained in studies involving O. sativa donors.

Tolerance to salinity is a complex polygenic trait linked to several morphological and physiological traits, with huge environmental influence and poor heritability (Gregorio and Senadhira, 1993; Flowers, 2004). Based on an earlier standard hydroponic system of screening (Gregorio et al., 1997) and the modified Yoshida culture-based method (Singh et al., 2010), there have been several investigations in screening the germplasm of O. sativa subspecies indica and japonica (Negrao et al., 2011) to identify donors. In rice, three major strategies, namely conventional breeding, molecular breeding, and genetic engineering, were employed to generate superior salinity-tolerant cultivars.

Wild rice species are treasure troves with various beneficial traits linked to yield, quality, and tolerance/resistance to abiotic and biotic stresses. Breeders often neglect the utilization of wild rice species for two main reasons. First, it is difficult to ensure gene flow from wild rice into the cultivated gene pool because of cross incompatibility, sterility, or non-viability in F₁ or backcrosses, restricted genetic recombination between wild and elite genomes, and linkage drag from wild rice. However, these genetic complications could be resolved with MABB, ensuring precise gene introgression with selections to minimize unwanted linkage drag and backcrosses, as compared to the traditional approach (Warburton et al., 2017). Second, when wild rice is grown outside their native habitat, either it is poorly acclimated or the expression of beneficial alleles is concealed. Ultimately, the performance of the derived introgression lines (ILs) is inferior.

Novel genes from the AA genome containing wild species in the primary gene pool could be easily transferred into domesticated rice through the traditional back-cross method. However, distant crosses between O. sativa and genetically remote wild species of the secondary and tertiary gene pools are difficult to achieve due to cross incompatibility and embryo abortion and/or degeneration. However, embryo rescue techniques can overcome these hurdles by producing distant fertile interspecific hybrids (Jena, 2010). Intergeneric hybrids between Porteresia coarctata, distant rice relatives, and O. sativa were produced with limited success by adopting vegetative multiplication of rescued hybrid embryos (Jena, 1994). Salt-tolerant genes can also be incorporated from O. porteresia into O. sativa through bridge crossing with O. australiensis (Latha et al., 2004; Mammadov et al., 2018).

With the latest advancements in NGS technology and high-throughput phenotyping, the historical natural genetic variation for salt tolerance present in a panel of wild rice accessions can easily be captured following GWAS. This is a potential strategy for mapping salt tolerance genes/QTLs, particularly in wild rice, where cross-incompatibility complicates the generation of mapping populations. The use of genetically distant wild species in the development of improved cultivars with superior trait performance has been greatly facilitated by the identification of candidate genes, GWAS, and development of introgression lines through MABB (Singh et al., 2021; Dai et al., 2022).

Transgenic approaches using cloned genes from wild species offer a solution to the issue of direct introgression of wild rice genes. Most transgenic methods have focused on single genes and/or one type of salt-tolerance mechanism in cultivars. However, genetic engineering utilizing cloned genes governing antioxidants, osmolytes, and ion transporters, photosynthesis enhancement, and yield simultaneously could potentially lead to the creation of more resilient rice varieties capable of thriving under high-salinity conditions.

The double haploid technique (DH) is another potential method for generating new homozygous salt-tolerant lines from distant crosses involving wild species in a single generation from heterozygous parents, which would otherwise require several generations of selfing to achieve near homozygosity in conventional breeding.

Genome editing is an emerging strategy for speeding up the development of advanced breeding lines, wherein a specific site in a target wild gene is edited directly by insertion, deletion, or alteration of existing nucleotide/s in the genomic segment of commercial varieties developed through distant hybridization. It corrects the deficiency, as in the present case, of salt sensitivity of the cultivars by avoiding laborious conventional or MABB and the incorporation of large introgressed genomic regions of wild species. Speed breeding is another promising approach for utilizing wild progenitors in breeding that manipulates the photoperiod and temperature to rapidly generate multiple generations within a year. In scenarios where precise genome editing is not an option, speed breeding plays a pivotal role in accelerating generation advancement, enabling selection against undesirable traits inherited from wild species, and stabilizing the genetic background of newly developed cultivars.

Although extensive studies have not been carried out in rice using wild species for genome editing and transgenic approaches, a considerable amount of research has been conducted on QTL mapping and introgression of QTLs/candidate genes in agronomically superior cultures.

Salinity, particularly during the reproductive stage, is a major abiotic stress factor that drastically reduces rice crop productivity. If not properly addressed, it poses a severe threat to global food security. Breeding for salinity tolerance is challenging due to its polygenic control, interaction with the environment, intricate physiological and metabolic processes, and growth stage. Despite the complexity, a moderate headway has been achieved with the development and release of approximately 101 salt-tolerant rice varieties using traditional global breeding methods.

The development of salt-tolerant cultivars has been hindered by the scarcity of genetic resources among domesticated cultivars. Therefore, future research should prioritize broadening the genetic base of modern cultivars by utilizing the salinity-adaptive genes present in unexplored wild species from secondary and tertiary gene pools. Prior to this endeavor, it is necessary to systematically explore and characterize various unexploited accessions of wild species for different salt stress defense mechanisms and identify genes for incorporation into breeding programs.

Obstacles associated with crossing and unwanted linkage drag are common during distant hybridization with wild species. This deficiency can be corrected by adopting the MABB method, which facilitates the accurate and accelerated introgression of target candidate genes/QTLs in popular cultivars. It is vital to reinforce pre-breeding programs that employ wild species in their breeding schemes worldwide. Continued research is needed to pinpoint more genomic resources in terms of salt-responsive QTLs and molecular markers and to profile the genes and their upstream regulatory regions in potential wild relatives. This will facilitate an in-depth understanding of the mechanisms that are effective in breeding tolerant varieties.

Exotic candidate genes have the potential to generate transgenic plants. The accurate pyramiding of genes responsible for different mechanisms of salt tolerance from divergent wild genetic sources into elite cultivars is needed for an hour to increase salt tolerance. Progress in transgenic and genome-editing techniques has paved the way for more possibilities to utilize and investigate valuable genes from wild rice to enhance salinity tolerance in cultivated rice.

The specific salt tolerance mechanisms in halophytes have yet to be fully elucidated. A comprehensive analysis of the process by which plants recognize salt stress and the crosstalk between different genes and pathways that are involved in regulating salt tolerance mechanisms is still needed. Concerted efforts are necessary to preserve both ex situ and in situ wild rice species for integration into salinity breeding programs.

GP: Conceptualization, Writing – original draft, Writing – review & editing. UB: Writing – review & editing. KR: Writing – review & editing. DB: Writing – review & editing. MA: Data curation, Writing – review & editing. RKS: Writing – review & editing. RMS: Writing – review & editing.